# US 11,028,377 B2 · Phytase variants YkAPPA having improved pepsin resistance and increased catalytic efficiency

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- Number: US 11,028,377 B2
- Document ID: US11028377B2
- Status: Granted
- Kind: B2
- Application number: 16/477,907
- Filed: 2018-01-15
- Published: 2020-09-03
- Granted: 2021-06-08
- Earliest claimed: 2017-01-15
- Assignees: Feed Research Institute Chinese Academy of Agricut
- Applicants: Feed Research Institute, Chinese Academy of Agricultural Sciences
- Inventors: Canfang Niu; Peilong Yang; Bin Yao; Yangyang Li; Dali Yu; Huiying Luo; Huoqing Huang; Yaru Wang
- Web page: https://patentgrep.com/patent/US11028377B2

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- C07K 14/24: from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C12Y 301/03: Phosphoric monoester hydrolases (3.1.3)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

The present invention relates to the field of genetic engineering, particularly to phytase variants YkAPPA having amino acid sequence substituting Leucine at the 162th site of the sequence set forth in SEQ ID NO.1 with glycine or proline, or having amino acid sequence substituting glutamic acid at the 230th site of the sequence set forth in SEQ ID NO.1 with glycine, alanine, serine, threonine, aspartic acid, proline, or arginine, and having improved pepsin resistance and increased catalytic efficiency of 2.1 times of that of the wild phytase, in the benefit of the development of economical feed enzyme industry.

## Claims

### Claim 1

A Phytase YkAPPA variant having the amino acid sequence obtained by substituting glutamic acid at the 230th site of the sequence of SEQ ID NO:1 with glycine, proline, or arginine, and having improved pepsin resistance and increased catalytic efficiency.

### Claim 2 (depends on 1)

A polynucleotide encoding the phytase YkAPPA variant of claim 1.

### Claim 3 (depends on 2)

The polynucleotide according to claim 2, wherein said polynucleotide is selected from the group consisting of SEQ ID NO:14, SEQ ID NO:19 or SEQ ID NO:20.

### Claim 4 (depends on 2)

A DNA construct comprising the polynucleotide of claim 2.

### Claim 5 (depends on 2)

A recombinant host cell comprising the polynucleotide of claim 2.

### Claim 6 (depends on 4)

A method of producing a phytase variant comprising the steps of: i.) transforming the host cell with the DNA construct of claim 4 to obtain a recombinant host cell; ii.) cultivating the recombinant host cell to produce the supernatant containing phytase variant; and iii.) recovering the said phytase variant.